Ca2+-independent insulin exocytosis induced by alpha-latrotoxin requires latrophilin, a G protein-coupled receptor.
alpha-Latrotoxin (alpha-LTX) induces exocytosis of small synaptic vesicles (SSVs) in neuronal cells both by a calcium-independent mechanism and by opening cation-permeable pores. Since the basic molecular events regulating exocytosis in neurons and endocrine cells may be similar, we have used the exocytosis of insulin-containing large dense core vesicles (LDCVs) as a model system. In primary pancreatic beta-cells and in the derived cell lines INS-1 and MIN6, alpha-LTX increased insulin release in the absence of extracellular calcium, but the insulin-secreting cell lines HIT-T15 and RINm5F were unresponsive. alpha-LTX did not alter membrane potential or cytosolic calcium, and its stimulatory effect on exocytosis was still observed in pre-permeabilized INS-1 cells kept at 0.1 microM Ca2+. Consequently, pore formation or ion fluxes induced by alpha-LTX could be excluded. The Ca2+-independent alpha-LTX-binding protein, latrophilin, is a novel member of the secretin family of G protein-coupled receptors (GPCR). Sensitivity to alpha-LTX correlated with expression of latrophilin, but not with synaptotagmin I or neurexin Ialpha expression. Moreover, transient expression of latrophilin in HIT-T15 cells conferred alpha-LTX-induced exocytosis. Our results indicate that direct stimulation of exocytosis by a GPCR mediates the Ca2+-independent effects of alpha-LTX in the absence of altered ion fluxes. Therefore, direct regulation by receptor-activated heterotrimeric G proteins constitutes an important feature of the endocrine exocytosis of insulin-containing LDCVs and may also apply to SSV exocytosis in neurons.
Lang
J
J
Division de Biochimie Clinique, Département de Médécine Interne, Centre Médical Universitaire, CH-1211 Genève 4, Switzerland. jochen.lang@medecine.unige.ch
Ushkaryov
Y
Y
Grasso
A
A
Wollheim
C B
CB
D000818:Animals; D001427:Bacterial Toxins / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology*; D002135:Calcium-Binding Proteins; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000502:physiology; D002463:Cell Membrane Permeability / Q000187:drug effects; D003600:Cytosol / Q000737:chemistry / Q000187:drug effects; D005089:Exocytosis / Q000187:drug effects* / Q000502:physiology; D019204:GTP-Binding Proteins / Q000378:metabolism; D015870:Gene Expression; D006023:Glycoproteins; D006460:Hemolysin Proteins / Q000494:pharmacology; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D007515:Islets of Langerhans / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000235:genetics; D008564:Membrane Potentials / Q000187:drug effects; D009419:Nerve Tissue Proteins / Q000235:genetics; D009479:Neuropeptides; D011485:Protein Binding; D011956:Receptors, Cell Surface / Q000378:metabolism; D018000:Receptors, Peptide / Q000235:genetics / Q000378:metabolism*; D013111:Spider Venoms / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D013211:Staphylococcus aureus / Q000737:chemistry; D050863:Synaptotagmin I; D050857:Synaptotagmins
D001427:Bacterial Toxins; D002135:Calcium-Binding Proteins; D006023:Glycoproteins; D006460:Hemolysin Proteins; D007328:Insulin; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; D009479:Neuropeptides; D011956:Receptors, Cell Surface; D018000:Receptors, Peptide; D013111:Spider Venoms; D050863:Synaptotagmin I; C034800:alpha-latrotoxin receptor; C112162:neurexophilin; C021091:staphylococcal alpha-toxin; D050857:Synaptotagmins; C086959:neurexin Ibeta; C020763:alpha-latrotoxin; D019204:GTP-Binding Proteins; D002118:Calcium